Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, and Penn State Hershey Cancer Institute, Hershey, Pennsylvania, USA.

Abstract

Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency.

DNMT1 expression is elevated during restricted EBV latency. Levels of DNMT1 within B-cell lines that maintain the EBV latency I, latency III, or Wp-restricted transcriptional program (Lat Prog) were assessed by immunoblot analysis. Ak-LCL is a B LCL generated by infection in vitro with the isolate of EBV obtained from the Akata BL line (Ak-BL); Reinf.Ak-BL is an EBV-negative (EBV-neg) Ak-BL cell line reinfected with a recombinant Akata isolate of EBV carrying a neomycin resistance gene in its BDLF3 gene (). β-Actin served as a loading control. N/A, not applicable.

DNMT3B expression is elevated during restricted EBV latency. The relative levels of the mRNAs encoding the de novo DNA methyltransferase DNMT3B during latency I and III maintained within paired Kem I/III and Mutu I/III BL cell lines were measured by qRT-PCR. Data shown are for the mRNA isoform 3B3 encoding DNMT3B () and are from a representative experiment in which each RNA/cDNA sample was analyzed in triplicate. GAPDH mRNA levels were used to normalize input between samples. The one-sample Student t test was used to determine statistical differences.

Reduction of DNMT1 levels in BL cells maintaining latency I does not result in reactivation of the latency III program. (A) Immunoblot analysis of DNMT1 expression in Kem I and Mutu I BL cell lines stably expressing control (Ctl.) or DNMT1-specific (shDNMT1) shRNAs. (B) EBV EBNA2 and LMP1 expression were assessed as markers of potential reactivation of the latency III program in the cell lines analyzed in panel A. Immunoblot detection of β-tubulin and β-actin served as loading controls in panels A and B, respectively.

Reduction of DNMT3B levels in BL cells maintaining latency I does not result in reactivation of the latency III program. (A) qRT-PCR analysis of DNMT3B in Kem I (left panel) and Mutu I (right panel) BL cell lines stably expressing control (Ctl.) or DNMT3B-specific (shDNMT3B) shRNAs. (B) EBV EBNA2 and LMP1 expression was assessed as markers of potential reactivation of the latency III program in the cell lines analyzed in panel A. Immunoblot detection of β-actin served as a loading control. The one-sample Student t test was used to determine statistical differences.

Combined knockdown of DNMT1 and DNMT3B does not reactivate latency III-associated mRNA and protein expression. (A) Demonstration by immunoblotting of the knockdown of DNMT1 within Kem I and Mutu I BL cell lines in which stable knockdown of DNMT3B had been previously achieved (), i.e., double knockdown (Dbl). Control (Ctl.) lines expressed the standard control (non-DNMT-specific) shRNA in addition to the DNMT3B-specific shRNA. EBV EBNA2 and LMP1 expression was assessed as markers of potential reactivation of the latency III program; immunoblot detection of β-tubulin and β-actin served as loading controls. (B) Lack of detection by RT-PCR of mRNAs from the latency III-specific EBNA promoters Cp and Wp indicates that transcriptional silencing of these promoters is sustained upon combined knockdown of DNMT1 and DNMT3B. Kem III and Mutu III served as positive controls for the detection of Cp and Wp usage; detection of Qp-specific EBNA1 mRNAs expressed during latency I in Kem I and Mutu I cells and their derivative cell lines served as a positive control for RNA integrity. Note that a faint larger cDNA amplified with Cp- and Wp-specific primers is the result of retention of the 81-bp intron between exons W1/W01 and W2. −RT, absence of reverse transcriptase in the cDNA synthesis reaction mixture prior to amplification by PCR.

Generation of ΔCTCF rEBV. Shown is the recombineering strategy used to generate a mutant rEBV genome deleted for the previously identified CTCF binding site within Cp (). (Left) Agarose gel electrophoresis of BamHI-digested and corresponding Southern blot of parental Ak-BAC-GFP DNA (lane 1), the intermediate BAC clone containing the targeting fragment with the cassette FRT-rpsL-tet-FRT in place of the CTCF binding site in the BamHI-C fragment of the EBV genome (lane 2), and the final BAC clone of ΔCTCF rEBV after Flp-mediated removal of the targeting cassette (lane 3). The black dot denotes the BamHI-C restriction fragment from the BAC clone of the Akata EBV genome (lane 1) and white dots the expected BamHI restriction fragments after insertion of the targeting cassette with a single BamHI restriction site in the tetracycline resistance gene into BamHI-C, resulting in loss of the BamHI-C fragment upon digestion (lane 2). The Southern blot was probed with 32P-labeled selection cassette fragment to ensure that inappropriate recombination had not occurred outside the desired locus. (Right) Configuration of the BamHI-C locus within the BAC clone of the Akata EBV genome is shown above the intermediate and final configurations of the locus during recombineering to delete the CTCF binding site upstream of Cp. The locations within BamHI-C of the Pol III genes encoding the EBER1 and EBER2 transcripts (small arrows), the latency origin of DNA replication oriP, the lytic cycle gene BCRF1 (dark block arrow), the CTCF binding locus, the EBNA2-responsive enhancer (open rectangle), and the Cp transcription start site (bent arrow) are indicated for reference. Vertical arrows indicate locations of BamHI restriction sites; BamHI restriction fragments denoted in lanes 1 and 2 in the ethidium bromide-stained agarose gel (left) are shown under the respective DNA configuration (right). After Flp-mediated removal of the FRT-flanked selection cassette, a single FRT site remains at the site of the 197-bp deletion that removed the CTCF binding site, resulting in a slightly smaller BamHI-C fragment.

Elimination of the CTCF binding site results in sustained usage of Cp. Kem I BL cells (latency I) were superinfected with either wt or ΔCTCF rEBV. (A) Detection of GFP expression indicates that superinfecting-virus genomes are retained. (B) Absence of EBNA2 and LMP1 (indicative of latency III; see Kem III positive control) suggests that CTCF is not essential for establishment of restricted latency. Detection of β-actin served as a loading control. (C) Analysis of EBNA promoter usage by RT-PCR. The low level of Cp-specific transcripts in cells superinfected with ΔCTCF but not wt rEBV suggests that CTCF is essential for efficient silencing of Cp. −RT, absence of reverse transcriptase in cDNA synthesis reaction mixture. All data shown were obtained from cells at approximately 12 months postsuperinfection.